Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.     

Germ cell chimerism in male marmosets

Marmosets normally produce biovular twins which are connected, via placental vascular anastomoses, as early as pre-somite stages of development. These anastomoses allow the exchange of lymphoid and hematopoietic tissue, as demonstrated by karyotype analysis in heterosexual twins. Because germ cells are also motile during development, i.e., they migrate from the yolk sac endodermal epithelium to the germinal ridges, the possibility exists that germ cells could also be exchanged between heterosexual twins. Testicular squash preparations were examined from 22 adult male marmosets of several species. During the diakinesis stage of meiotic prophase the XY chromosome pair was distinct. Spermatocytes which lacked the conspicuous end-to-end association of the XY pair were considered to have originated from germ cells with an XX sex chromosome constitution. In approximately half the animals examined, all diakinesis figures contained an end-to-end XY pair. In the remaining animals, primary spermatocytes were seen that clearly did not contain an endto-end XY pair. The largest number of such XX primary spermatocytes in any one animal was 21% (9 of 43 cells examined). The effects of germ cell chimerism on the sex ratio of offspring was also investigated.     

Germ cell loss in the XXY male mouse: Altered X-chromosome dosage affects prenatal development

Male mammals with two X chromosomes are sterile due to the demise of virtually all germ cells; however, the underlying reasons for the germ cell loss remain unclear. The use of a breeding scheme for the production of XXY male mice has allowed us to experimentally address the question of when and why germ cells die in the XXY testis and whether the defect is due to the presence of an additional X chromosome in the soma, the germ cells themselves, or both. Our studies demonstrate that altered X-chromosome dosage acts to impair germ cell development in the testis at a much earlier stage than suggested by previous studies of XX sex-reversed males or XX/XY chimeras. Specifically, we noted significantly reduced germ cell numbers in the XXY testis during the period of germ cell proliferation in the early stages of testis differentiation. Although the somatic development of the XXY testis is morphologically and temporally normal, our studies indicate that germ cell demise reflects a defect in somatic/germ cell communication, since, in an in vitro system, the proliferative potential of fetal germ cells from XXY males is indistinguishable from that of normal males. Mol. Reprod. Dev. 49:101–111, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.     

Marker Chromosome Analysis of Tetraparental AKR↔CBA-T6 Mouse Chimaeras

Marker chromosome analysis of 18 tetraparental AKR↔CBA/H-T6 chimaeras revealed a great excess of AKR mitoses over CBA mitoses in direct preparations from lymphomyeloid tissues, corneal epithelium, intestinal epithelium and skin (Table 1). The degree of AKR dominance was strongly influenced by anatomical site (Tables 2 and 3). The testes of three out of four XY/XY males contained a marked excess of AKR germ cells and produced a parallel excess of functional AKR gametes (Table 4). Mitotic spreads in mitogen-stimulated cultures of tail blood were overwhelmingly of AKR type in 1972, but less so in 1973 (Table 5).
The coat phenotypes, and breeding results from known or presumptive XX/XX females, suggest that AKR and CBA cells were numerically balanced when melanoblasts and oocytes were formed during embryonic development, and therefore that the striking deviations from equality observed in mitotic populations of adult chimaeras arose later by differential proliferation or survival of AKR cells, or both.
The low frequency of lymphomas, compared with normal AKR mice, previously reported in these chimaeras cannot therefore be accounted for by insufficiency of AKR cells in the thymus or elsewhere in the lymphomyeloid tissues. One of three lymphomas studied was CBA type. This suggests that the high risk of lymphomatous transformation is not an autonomous property of AKR cells.     

60,XY/60,XX chimerism in the germ cell line of mature bulls born in heterosexual twinning

According to present knowledge there is a germ cell chimerism (XY/XX) in young bulls born in heterosexual twinning due to exchange of primordial germ cells in embryonic life. These germ cells were believed to have been eliminated in the young bull. Two-color fluorescence in situ hybridization (FISH) identification of the sex chromosomes by biotinylated and digoxygenin labeled probes have been used. The material consisted of three bulls born in heterosexual twinning. The results obtained indicated that even mature bulls (more than two years old) demonstrate spermatogonial chimerism. Several authors state that the bulls with blood cell chimerism, originating from dizygous twinning, are characterized by decreased fertility. Changes of the sex ratio of offspring due to proliferation of the female cells have also been proposed. The present observations should give a renewed interest in checking the possibility of survival and differentiation of germ cells from the female partner in the germ cell lines.     

X inactivation in a mammal species with three sex chromosomes

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

Detection of leucochimaerism in bovine twins by DNA fingerprinting

Karyotyping and hypervariable genetic markers indicate extensive leucochimaerism between pairs of dizygotic twins in cattle, a result of placental vascular anastomosis. The extent of this chimaerism includes both kind and number of cells exchanged. All heterosexual twin pairs harboured two types of leucocytes, having either XX or XY chromosome pairs, and 30 of 31 pairs of twins shared identical DNA fingerprints. Although chromosome results from skin fibroblasts indicate that some chimaerism occurs in the skin, the low level allows for differentiation of genotypes between twins. The results warrant against the common practice of using blood samples for DNA typing if twinning is not properly documented.     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

Ontogeny of X-chromosome inactivation in the female germ line

Data are presented on G6PD electrophoretic patterns in fetal ovarian preparations of G6PD heterozygotes. The results indicate that in the early or mitotic period of female germ cell development, only a single X chromosome is active in each oogonium just as is the case for X inactivated somatic tissue. However, in the later or meiotic stage, reactivation of the inactive X chromosome in each oocyte occurs so that two functional X chromosomes are present in each oocyte.     

XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima

We show that the geophilomorph centipede Strigamia maritima possesses an XX/XY system of sex chromosomes, with males being the heterogametic sex. This is, to our knowledge, the first report of sex chromosomes in any geophilomorph centipede. Using the recently assembled Strigamia genome sequence, we identified a set of scaffolds differentially represented in male and female DNA sequence. Using quantitative real-time PCR, we confirmed that three candidate X chromosome-derived scaffolds are present at approximately twice the copy number in females as in males. Furthermore, we confirmed that six candidate Y chromosome-derived scaffolds contain male-specific sequences. Finally, using this molecular information, we designed an X chromosome-specific DNA probe and performed fluorescent in situ hybridization against mitotic and meiotic chromosome spreads to identify the Strigamia XY sex-chromosome pair cytologically. We found that the X and Y chromosomes are recognizably different in size during the early pachytene stage of meiosis, and exhibit incomplete and delayed pairing.     

Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

Meiotic chromosomes of the spermatocytes ejaculated by bulls

The Sperling and Kaden (1971) approach was used in studies of the bull meiotic chromosomes. The number of meiocytes in the ejaculates of normal bulls ranged from 0.0001 to 0.0114% of the whole set of cellular elements in the ejaculate. Two of the studied bulls had a chromosomal abnormality in somatic cells (60, XX/60, XY mosaic). These bulls displayed no deviation as concerns the quantity of meiocytes in their ejaculates. In two other bulls examined, with cryptorchism and azoospermia, about 0.0017 and 4.75% meiocytes were counted in the ejaculate, respectively. A comparative analysis of ejaculated chromosomes and meiotic chromosomes obtained from the normal testes by biopsy did not reveal any differences in the chromosomal morphology. Various premeiotic and meiotic stages, from spermatogonium A and B to metaphase 1, were identified in the bull ejaculates. This technique may be valuable in cytogenetic diagnosis of meiotic abnormalities in sires.     

Accidental X-Y recombination and the aetiology of XX males and true hermaphrodites

Accidental recombination between the differential segments of the X and Y chromosomes in man occasionally allows transfer of Y-linked sequences to the X chromosome leading to testis differentiation in so-called XX males. Loss of the same sequences by X-Y interchange allows female differentiation in a small proportion of individuals with XY gonadal dysgenesis. A candidate gene responsible for primary sex determination has recently been cloned from within this part of the Y chromosome by Page and his colleagues. The observation that a homologue of this gene is present on the short arm of the X chromosome and is subject to X-inactivation, raises the intriguing possibility that sex determination in man is a quantitative trait. Males have two active doses of the gonad determining gene, and females have one dose. This hypothesis has been tested in a series of XX males, XY females and XX true hermaphrodites by using a genomic probe, CMPXY1, obtained by probing a Y-specific DNA library with synthetic oligonucleotides based on the predicted amino-acid sequence of the sex-determining protein. The findings in most cases are consistent with the hypothesis of homologous gonad-determining genes, GDX and GDY, carried by the X and Y chromosomes respectively. It is postulated that in sporadic or familial XX true hermaphrodites one of the GDX loci escapes X-inactivation because of mutation or chromosomal rearrangement, resulting in mosaicism for testis and ovary-determining cell lines in somatic cells. Y-negative XX males belong to the same clinical spectrum as XX true hermaphrodites, and gonadal dysgenesis in some XY females may be due to sporadic or familial mutations of GDX.     

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.